GSM7179302: HCT116 cells, chromatin RNA, untreated condition, rep3; Homo sapiens; OTHER
1 x 10^7 HCT116 cells were harvested with trypsin after 8hrs of treatment with 1mM of HU, or PBS and washed with ice-cold PBS. One tenth aliquot of cells was put aside for total RNA isolation, the remaining was used for cell fractionation, according to[2]. All steps were performed in presence of protease inhibitors (Roche), phosphatase inhibitors (Roche) and RNAsin (Promega). Cells were resuspended in 200 µl of isotonic lysis buffer buffer (10mM Tris-Hcl pH7, 150 mM NaCl, 0.15% NP-40) for 5 min on ice and layered on a sucrose buffer (10mM Tris- HCl, 150 mM NaCl, 25% sucrose). Nuclei were centrifuged for 10 min at full speed to recover the supernatant as the cytoplasmic fraction. The nuclear pellet was washed (1 mM EDTA, 0.1% Triton-X100 in PBS), and resuspended in 200 µl glycerol buffer (20mM Tris-HCl pH8, 75mM NaCl, 0.5 mM EDTA, 505 glycerol, 0.85 mM DTT) and finally lysed with 200 µl of nuclear lysis buffer (20mM HEPES, 300mM NaCl, 1M urea, 0,2 mM EDTA, 1% NP-40, 1mM DTT). Lysed nuclei were centrifuged at full speed for 2 min to separate the soluble fraction (supernatant) from the chromatin associated fraction (pellet). RNA was isolated and DNAse I-treated with Maxwell RSC Simply RNA kit (Promega) RNA was processed for library preparation with TruSeq Stranded Total RNA kit (Illumina), according to the manufacturer's instructions